新牧1号苜蓿两种抗逆相关启动子的功能分析

前期已成功克隆了新牧1号苜蓿MvP5CS与MvNHX1基因和二者的启动子序列。在此基础上,本试验分别构建了含有Mvp5cs和Mvnhx1启动子的调控GUS报告基因的植物表达载体,并通过农杆菌介导法得到了含有Mvnhx1与Mvp5cs启动子的转基因烟草植株。PCR检测证明了两种启动子已稳定的整合到烟草基因组中;GUS组织染色证明两种启动子均具有启动基因表达的功能。通过测量GUS活性来探究两种不同的诱导型启动子在4种非生物胁迫下(干旱、盐、脱落酸、赤霉素)的表达活性。结果表明,1)Mvnhx1与Mvp5cs启动子均响应盐、脱落酸、赤霉素、干旱胁迫,与CaMV35S启动子相比,差异显著。2)Mvnhx1启动子在盐胁迫、脱落酸胁迫下所起诱导作用要优于Mvp5cs启动子。在48h100mmol/L、150mmol/L NaCl胁迫处理时,Mvnhx1启动子GUS活性分别是Mvp5cs启动子的2.03和3.23倍,差异显著。在25μmol/L、50μmol/L ABA处理下,Mvnhx1启动子的活性整体高于另两种启动子,差异显著。3)Mvp5cs启动子在干旱胁迫、赤霉素胁迫所起诱导作用优于Mvnhx1启动子。干旱胁迫时,Mvp5cs启动子的GUS活性在36h是同时间Mvnhx1启动子的2.22倍。70μmol/L GA处理时,Mvp5cs启动子在36h达到最大值,是同时间段Mvnhx1启动子的1.79倍。

Functional analysis of two stress-related promoters in Medicago varia cultivar Xinmu No.1

MvP 5CS and MvNHX1 genes of the Medicago varia cultivar Xinmu No.1 and their promoter se-quences have been successfully cloned in previous work.Based on this research,in this study two plant expres-sion vectors,each containing Mvp5cs and Mvnhx1 promoters with a GUS gene as the reporter,were construc-ted and two tobacco transgenic plants with the promoter obtained by Agrobacterium-mediated method.PCR a-nalysis showed that the two promoters had been stably integrated into the tobacco genome and GUS-staining proved that they have the function of activating gene expression.We explored this function by measuring GUS activity under four different abiotic stresses:drought,salinity,abscisic acid (ABA)and gibberellin (GA).The results showed that Mvnhx1 and Mvp5cs responded to the four stresses and had significant differences from the CaMV35S promoter.The Mvnhx1 promoter was superior to Mvp5cs under salt and ABA stress.After 48 h treatments of 100 and 150 mmol/L NaCl stress,the GUS activity of Mvnhx1 was 2.03 times and 3.23 times that of Mvp5cs .Under treatments of 25 and 50 μmol/L ABA stress,Mvnhx1 activity was significantly higher than the other two promoters.Mvp5cs was superior to Mvnhx1 under drought and GA stress.The GUS activi-ty of the Mvp5cs was 2.22 times that of Mvnhx1 after 36 h treatment of drought stress.The Mvp5cs promoter reached its maximum value,which was 1.79 times that of Mvnhx1,after 36 h treatment with 70 μmol/L GA stress.